Hydroxysteroid dehydrogenases (HSDs) play pivotal roles in the biosynthesis and inactivation of all steroid hormones. In target tissues they regulate occupancy of nuclear receptors by interconverting potent steroid hormones with their cognate inactive metabolites. HSDs belong to two protein superfamilies the short-chain dehydrogenase/ reductases (SDRs) and the aldo-keto reductases (AKRs). Rat liver 3alpha-HSD is the most thoroughly characterized HSD that is an AKR. Crystal structures of the apoenzyme [E], its binary complex [E NADP+] and ternary complex with a competitive inhibitor [E NADP+ testosterone] are described. Structure-function studies on 3alpha-HSD will provide unique insight into catalysis and ligand recognition in all steroid metabolizing AKRs (e.g., 3alpha-, 17beta-, and 20alpha-HSDs, and delta 4-3 ketosteriod 5beta-reductase). X-ray crystal structures of recombinant rat liver 3alpha-HSD (rr3alpha- HSD) containing either 5alpha-dihydrotestosterone (5alpha-DHT) or 5beta- DHT are now sought. In these structures the 3-ketosteroid substrate is either planar (A/B trans-ring fusion) or significantly bent (A/B cis- ring fusion) and will identify the catalytic acid. The structure of recombinant human type 2 3alpha-HSD NADP+ 4-androstene-3, 17-dione complex is also sought. This AKR has both 3alpha- and 17beta-HSD activity, may regulate prostate androgen receptor occupancy, yet binds steroids backwards (D ring instead of A ring first) and upside down (alpha-face in the beta-face orientation). Using rr3alpha-HSD, stopped- flow fluorescence spectroscopy, primary deuterium (4R-NAD(P)D), and solvent (D20) kinetic isotope effects will determine whether movement of a nucleotide-clamping loop is rate-limiting in the kinetic mechanism, and whether hydride transfer or proton donation is rate-limiting in the chemical step. pH rate profiles using mutants of the catalytic tetrad (Y55, H117, K84 and D50) will reveal the identity of the general acid by titration. Residues involved in cofactor binding will be mutated to either invert the stereochemistry of hydride transfer or change preference from NADPH to NADH. Knowledge of the steroid pocket will be exploited to alter specificity: (1) 5beta-reductase activity will be introduced by mutating tetrad residues; (2) 20alpha-HSD activity will be engineered by either mutating residues in the steroid pocket or by constructing 3alpha/20alpha-HSD chimeras in which loop regions of the pocket are swapped; and (4) the C-terminal loop (a major determinant of 3alpha- 17beta- and 20alpha-HSD specificity) will be randomly mutagenized by phage-display.